PROJECT SUMMARY Targeted therapies are based on diseased cells having characteristics that distinguish them from healthy cells. Several pathological states including cancer, ischemia, and inflammation are characterized by acidosis. We propose to exploit acidosis to develop new technologies for targeted therapies. We have recently developed new peptides that insert into membranes only in acidic conditions. Preliminary data show that these acidity-triggered rational membrane (ATRAM) peptides have favorable biophysical properties and target cells in a pH-responsive fashion. We will determine the optimal peptide sequence in the context of the transverse asymmetry in lipid composition found in most cellular membranes. Next, we will determine the pKa of the residues responsible for triggering membrane insertion under acidic conditions. This will be instrumental in elucidating the molecular mechanism of ATRAM peptides. We have evolved the design of the ATRAM peptides to design bifunctional peptides that have the potential to modulate the activity of the EphA2 receptor. EphA2 is involved in mediating invasion and metastasis in different tumor types. Our data suggest that these peptides are soluble, but are also able to insert into membranes and activate the EphA2 receptor. We will study peptide control over the clustering of the EphA2 receptor. A set of peptides of different sequences will be used as a tool to determine the conformational changes associated to the formation of the membrane clusters of EphA2, leading to receptor activation. This multidisciplinary study will lead to a molecular understanding of the mode of action of these peptides. This knowledge will provide important clues to develop future therapeutic technologies that specifically target the acidosis of diseased tissues.